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Recombinant Proteins & Enzymes

Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated

Catalog No: 31555 Format: 20 µg $445 Buy Now
Expressed In: E. coli Protein Species: Human

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Expressed Protein Ligation

Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated has been generated using the patented Expressed Protein Ligation (EPL) technology. EPL can be used to generate methylated, acetylated or phosphorylated histones. In EPL, the histone globular domain is ligated to a peptide that contains the N-terminal histone tail with the desired site-specific modification. The ligation reaction maintains the native histone bonds, and the resulting recombinant protein more closely mimics natural histones.

for Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylatedContents

Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated (20 µg protein + 20 µg DNA) are supplied in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM DTT and 20% glycerol. Please refer to product insert upon arrival for lot-specific concentration.

for Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylatedBackground

In vivo, histones are wrapped around by DNA in chromatin. Therefore, nucleosomes are more physiologically relevant substrates than histones and histone-derived peptides for in vitro studies. More importantly, some histone methyltransferases are significantly more active, as well as specific, when using nucleosomal substrates in Histone Methyltransferase (HMT) assays, such as DOT1L and NSD family enzymes. Nucleosomes are also widely used in histone methyltransferase screening assays to identify small molecular inhibitors for drug discovery.

for Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylatedApplication Notes

Recombinant Nucleosome H3K9me3 (EPL) is suitable for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling.

for Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylatedProtein Details

Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated consist of a 167 bp of 601 DNA with 5’ biotin tag and two molecules each of histones H2A that includes amino acids 1-130 (end) (accession number NP_003503.1), H2B that includes amino acids 1-126 (end) (accession number NP_003509.1), H3.2 that includes amino acids 1-136 (end) (accession number NP_066403.2) with trimethylation at lysine 9, and H4 that includes amino acids 1-103 (end) (accession number NP_003539.1). All of these histones were expressed in E. coli cells. The molecular weight of histone octamer is ~108 kDa. The recombinant protein is ≥95% by SDS-PAGE gel. H3K9me3 (Histone H3 trimethyl Lys9) protein is generated using expressed protein ligation (EPL) technology. Truncated human Histone H3.2 is produced in E. coli and purified using FPLC. The purified protein is subsequently ligated to a N-terminal histone tail peptide containing trimethyl lysine 9 via a native peptide bond.

 
Streptavidin pull-down for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.

Streptavidin pull-down for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.
Recombinant Mononucleosomes H3K9me3 (EPL) - biotin were pulled down by streptavidin beads. Input nucleosomes (Lane 2) and the nucleosomes (Lane 3) pulled down by streptavidin were run on a 13% SDS-PAGE gel and stained with Commassie blue. The SDS-PAGE gel result shows that almost all of biotinylated mononucleosomes H3K9me3 are pulled down by streptavidin beads. * indicates streptavidin.

Recombinant Mononucleosomes H3K9me3 (EPL) - biotin, DNA gel.

Recombinant Mononucleosomes H3K9me3 (EPL) - biotin, DNA gel.
Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated were run on a 2% agarose gel and stained with ethidium bromide. Lane 1: DNA marker. Lane 2: Intact monnucleosomes H3K9me3. Lane 3: 601 DNA. Intact mononucleosomes H3K9me3 migrated much higher than free 601 DNA. The agarose gel result shows almost all of 601 DNA wrap histone octamers to form nucleosomes.

Western Blot analysis for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.

Western Blot analysis for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.
Unmodified nucleosomes (Lane 1) and Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated (Lane 2) were detected with anti-H3K9me3 antibody and anti-H4 antibody, respectively. H4 was detected as loading control. Only Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated can be detected by anti-H3K9me3 antibody.

for Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylatedStorage

Recombinant proteins in solution are temperature sensitive and must be stored at -80°C to prevent degradation. Avoid repeated freeze/thaw cycles and keep on ice when not in storage.

for Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylatedGuarantee

This product is guaranteed for 6 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

 

Technical Data Sheet

Recombinant Mononucleosomes H3K9me3 (EPL) - biotinylated
 

Data Thumbnails

Streptavidin pull-down for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.

Streptavidin pull-down for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.

Recombinant Mononucleosomes H3K9me3 (EPL) - biotin, DNA gel.

Recombinant Mononucleosomes H3K9me3 (EPL) - biotin, DNA gel.
(Click to enlarge and view details).

Western Blot analysis for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.

Western Blot analysis for Recombinant Mononucleosomes H3K9me3 (EPL) - biotin.
(Click to enlarge and view details).